SUMMARY Although the publication of a human genome blueprint occurred more than 15 years ago, we remain far from understanding the complexities of the human proteome ? the collections of proteins and their interactions that define individual human cells. The complexity of the proteome is both immense and dynamic, reflecting diverse, context-specific expression of genes in individual cells and distinct isoforms for individual proteins. In addition, individual proteins may participate in several distinct protein assemblies during their lifetime and undergo dynamic signal-dependent re-organization in order to impart distinct functions and cellular attributes. Moreover, numerous protein assemblies self-combine and compartmentalize to generate organelles and signaling modules within the cell, which are inherently dynamic. During the past 5 years, we have designed, validated, and applied a platform for the large scale analysis of protein interaction partners using affinity purification-mass spectrometry (AP-MS) termed BioPlex, which has allowed us to profile interaction partners for 10,000 nonredundant human bait proteins in HEK293T. In total, an atlas of nearly 120,000 protein-protein interactions was identified. The majority have not been reported through independent efforts. The robustness of BioPlex, when benchmarked against other studies as well as our initial analysis of a similar effort in another cell line (HCT116), parallels or exceeds available resources, allowing us to broadly define human protein communities, predict functions and localizations of unstudied proteins based on interaction partners, and define a large number of domain-domain enrichments that begin to impart structural architecture upon the network. In this renewal, we seek to greatly enhance and extend these efforts in three major ways: First, we will complete a full-pass, 10K-bait interactome in HCT116 cells and further address cell type diversity in interactomes by performing an analysis of 2000 high-priority bait proteins in 4 or more additional cell lines. Second, in order to systematically address the complexities of membrane protein interactomes, we will perform proximity labeling (biotinylation) across a broad range of 500 proteins known or predicted to reside in cellular membranes, more than half of which lack sub-cellular locations in UNIPROT. This will begin to provide a global understanding of membrane protein assemblies and will help to define the spatial architecture of the proteome. Third, we will develop a platform for quantitative analysis of the interactomes of 200 high-priority, mutant disease genes, thereby providing an in-depth view of how mutations drive reorganization of key networks. Together, these studies will: i) define interactomes across multiple cell types, ii) provide a spatial view of membrane protein architecture, and iii) begin to globally define how mutant alleles short-circuit key cellular systems to promote disease. 1